The purpose of the proposed study is to elucidate the molecular mechanism of catecholamine stimulated non-shivering thermogenesis. Both skeletal muscle and brown adipose tissue have been implicated as largely responsible for the 2-3 fold increases in O2 consumption observed in cold-adapted rats given catecholamines or exposed to low ambient temperatures. In the present study the perfused rat hemicorpus will be used to study skeletal muscle metabolism and isolated brown fat adipocytes to study the brown adipose tissue metabolism. Rats will be cold-adapted by exposing them to 4 degrees C ambient temperatures for 3-4 weeks prior to use. An initial goal will be to determine whether increased rates of tissue respiration which occur in the presence of noradrenalin or isoproterenol result from increased substrate delivery, increases in cytosolic ADP due to increases in sodium pump activity or uncoupling of mitochondrial O2 uptake from phosphorylation. The approach used will be (1) to measure sodium pump activity isotopically by loading the tissue with 22Na ion and then measuring efflux rates (2) to vary substrate delivery to the tissues by artificial means and (3) to use certain inhibitors such as oligomycin, an inhibitor of mitochondrial ATP-ase, and bongkrekic acid, an inhibitor of mitochondrial ADP transport, to block the mitochondria response to cytosolic ADP. The study is unique and may provide some new perspectives on the mechanism of non-shivering thermogenesis because it employs an adequate model of muscle perfusion which will permit both measurement of tissue metabolite levels as well as the manipulation of the muscle environment with respect to both substrate and hormone availability. The studies of brown fat are likewise unique because new techniques of rapid cell fractionation will be used to determine compartmentation of critical metabolites between cytosol and mitochondria.